Method for chemoprevention of prostate cancer

ABSTRACT

This invention provides the chemoprevention of prostate cancer and, more particularly, to a method of preventing prostate carcinogenesis comprising the steps of administering to a human subject having a precancerous precursor of prostate adenocarcinoma, a pharmaceutical preparation comprising a chemopreventive agent to prevent, prevent recurrence of, suppress or inhibit prostate carcinogenesis. The present invention provides a safe and effective method for suppressing or inhibiting latent prostate cancer and is particularly useful for treating subjects having elevated risk of developing prostate cancer, for example, those having benign prostatic hyperplasia, prostate intraepithelial neoplasia (PIN), or an abnormally high level of circulating prostate specific antibody (PSA), or who have a family history of prostate cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part application of U.S. Ser. No.09/531,472, filed Mar. 20, 2000, which is a Continuation-in-Partapplication of U.S. Ser. No. 09/436,208, filed Nov. 8, 1999, which is aContinuation-in-Part application of U.S. Ser. No. 09/306,958, filed May7, 1999, which claims priority of United States Provisional ApplicationNo. 60/084,602, filed May 7, 1999 which is hereby incorporated byreference in its entirety.

FIELD OF INVENTION

This invention relates to the chemoprevention of prostate cancer and,more particularly, to a method of suppressing or inhibiting latentprostate cancer comprising administering to a mammalian subject having aprecancerous precursor of prostate adenocarcinoma, a pharmaceuticalpreparation comprising a chemopreventive agent and analogs andmetabolites thereof. The chemopreventive agent prevents, preventsrecurrence of, suppresses or inhibit prostate carcinogenesis.

BACKGROUND OF THE INVENTION

Prostate cancer is one of the most frequently occurring cancers amongmen in the United States, with hundreds of thousands of new casesdiagnosed each year. Unfortunately, over sixty percent of newlydiagnosed cases of prostate cancer are found to be pathologicallyadvanced, with no cure and a dismal prognosis. One approach to thisproblem is to find prostate cancer earlier through screening programsand thereby reduce the number of advanced prostate cancer patents.Another strategy, however, is to develop drugs to prevent prostatecancer. One third of all men over 50 years of age have a latent form ofprostate cancer that may be activated into the life-threatening clinicalprostate cancer form. The frequency of latent prostatic tumors has beenshown to increase substantially with each decade of life from the 50s(5.3-14%) to the 90s (40-80%). The number of people with latent prostatecancer Is the same across, all cultures, ethnic groups, and races, yetthe frequency of clinically aggressive cancer is markedly different.This suggests that environmental factors may play a role in activatinglatent prostate cancer. Thus, the development of chemopreventionstrategies against prostate cancer may have the greatest overall impactboth medically and economically against prostate cancer.

Because of the high incidence and mortality of prostate cancer, it isimperative to develop chemoprevention strategies against thisdevastating disease. Understanding those factors that contribute toprostate carcinogenesis including the initiation clues as theappropriate points of intervention to prevent molecular mechanisticclues as to appropriate points of intervention to prevent or halt thecarcinogenic process. New innovative approaches are urgently needed atboth the basic science and clinical levels to decrease the incidence ofprostate cancer as well as to haft or cause the regression of latentprostate cancer. As the frequency of prostate cancer escalatesdramatically at the same ages when men are confronted by other competingcauses of mortality, simply slowing the progression of prostateadenocarcinoma may be both a more suitable and cost effective healthstrategy.

Various approaches have been taken to the chemoprevention of prostatecancer. Greenwald, Expanding Horizons in Breast and Prostate CancerPrevention and Early Detection in J. Cancer Education, 1993, Vol. 8, No.2, pages 91-107, discusses the testing of 5α-reductase inhibitors suchas finasteride for the prevention of prostate cancer. Brawley et al.,Chemoprevention of Prostate Cancer in Urology, 1994, Vol. 43. No. 5,also mentions 5α-reductase inhibitors as well as difluoromethylornithineand retinoids as potential chemopreventive agents.

Kelloff et al., Introductory Remarks: Development of ChemopreventiveAgents for Prostate Cancer in Journal of Cellular Biochemistry, 1992,Supplement 16H: 1-8, describes National Cancer Institute preclinicalstudies of seven agents: all-trans-N-(4-hydroxyphenyl)retinamide,difluoromethylornithine, dehydroepiandrosterone, liarozole, lovestatin,oltipraz, and finasteride.

Lucia et al., Chemopreventive Activity of Tamoxifen,N-(4-Hydroxyphenyl)retinamide, and the Vitamin D Analogue Ro24-553 1 forAndrogen-promoted Carcinomas of the Rat Seminal Vesicle and Prostate @in Cancer Research, 1995, Vol. 55, Vol. 55, pages 5621-5627, reportschemoprevention of prostate carcinomas in Lobund-Wjstar rats bytamoxifen, and estrogen response modifier.

As discussed in Potter et al., A mechanistic hypothesis for DNA adductformation by tamoxifen following hepatic oxidative metabolism inCarcinogenesis, 1994, Vol. 15, No. 3, pages 439-442, tamoxifen causesliver carcinogenicity in rats, which is attributed to the formation ofcovalent DNA adducts. This reference also reports that the tamoxifenanalogue toremifene, which showed a much lower level of hepatic DNAadduct formation than tamoxifen, is non-carcinogenic.

Toremifene is an example of a triphenylalkene compound described In U.S.Pat. Nos. 4,696,949 and 5,491,173 to Toivola et al., the disclosures ofwhich are incorporated herein by reference. The parenteral and topicaladministration to mammalian subjects of formulations containingtoremifene are described in U.S. Pat. No. 5,571,534 to Jalonen et al.and in U.S. Pat. No. 5,605,700 to DeGregorio et al., the disclosures ofwhich are incorporated herein by reference.

Toremifene-containing formulations for reversing the multidrugresistance to cancer cells to a cytotoxic drug are described in U.S.Pat. No. 4,990,538 to Harris et al., the disclosure of which isincorporated herein by reference. U.S. Patent Nos. 5,595,722 and5,599,844 to Grainger et al., the disclosures of which are incorporatedherein by reference, describe methods for identifying agents thatincrease TGFP levels and for orally administering formulationscontaining TGFP activators and TGFP production stimulators to prevent ortreat conditions characterized by abnormal proliferation of smoothmuscle cells, for example, vascular trauma. Disclosed agents forincreasing TGFP levels include tamoxifen and its analogue toremifene.

U.S. Pat. Nos. 5,629,007 and 5,635,197 to Audia et al., the disclosuresof which are incorporated herein by reference, describe a method ofpreventing the development of prostatic cancer at risk of developingsuch cancer, for example, a patient having benign prostatic hyperplasia,by administering to the patient an octahydrobenzo[f}quinolin-3-onecompound.

U.S. Pat. No. 5,595,985 to Labrie, to disclosure of which isincorporated herein by reference, also describe a method for treatingbenign prostatic hyperplasia using a combination of a 5α-reductaseinhibitor and a compound that binds and blocks access to androgenreceptors. One example of a compound that blocks androgen receptors isflutamide.

U.S. Pat. Nos. 4,329,364 and 4,474,813 to Neri et al., the disclosuresof which are incorporated herein by reference, describe pharmaceuticalpreparations comprising flutamide for delaying and/or preventing theonset of prostate carcinoma. The preparation can be in the form of acapsule, tablet, suppository, or elixir. Despite these developments,there is a continuing need for agents and methods effective forpreventing prostate cancer. The present invention is directed tosatisfying this need.

SUMMARY OF THE INVENTION

This invention relates to the chemoprevention of prostate cancer and,more particularly, to a method of suppressing or inhibiting latentprostate cancer comprising the steps of administering to a mammaliansubject a pharmaceutical preparation comprising a chemopreventive agentand analogs, and metabolites thereof.

This invention relates to the chemoprevention of prostate cancer and,more particularly, to a method of suppressing or inhibiting latentprostate cancer comprising the steps of: administering to a mammaliansubject having a precancerous precursor of prostate adenocarcinoma, apharmaceutical preparation comprising a chemopreventive agent andanalogs, and metabolites thereof.

This invention provides a method of administering to a subject aneffective dose of an antiestrogen which does not cause the formation ofDNA adducts, to prevent, prevent recurrence of, and/or suppress orinhibit latent prostate cancer or prostate carcinogenesis.

This invention provides a method of suppressing or inhibiting latentprostate cancer comprising the steps of: administering to a subject aneffective dose of a chemopreventive agent, toremifene and analogs ormetabolites thereof, to prevent, prevent recurrence of, and/or suppressor inhibit prostate carcinogenesis.

The present invention is directed to a method of suppressing orinhibiting latent prostate cancer comprising the steps of: administeringto a mammalian subject having a precancerous precursor of prostateadenocarcinoma and does not have prostate cancer, a pharmaceuticalpreparation comprising a chemopreventive agent having the formula:

wherein R₁ and R₂, which can be the same or different, are H or OH, R₃is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different,are H or an alkyl group of 1 to about 4 carbon atoms and analogs, andmetabolites thereof; and their pharmaceutically acceptable carrier,diluents, salts, esters, or N-oxides, and mixtures thereof. In oneembodiment the precancerous precursor of prostate adenocarcinoma isprostate intraepithelial neoplasia (PIN). In one embodiment theprecancerous precursor of prostate adenocarcinoma is high grade prostateintraepithelial neoplasia (PIN).

The present invention provides a safe and effective method forpreventing prostate carcinogenesis, suppressing or inhibiting latentprostate cancer and is particularly useful for treating subjects havingand elevated risk of developing prostate cancer, for example, thosehaving benign prostatic hyperplasia, prostate intraepithelial neoplasia(PIN), or an abnormally high level of circulating prostate specificantibody (PSA), or who have a family history of prostate cancer,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A graph illustrating the chemopreventive effects of toremifenein the TRAMP model.

FIGS. 2A-2C: H&E sections illustrating ventral prostate cells in normalmice and prostate carcinoma in TRAMP mice included in the study.

FIG. 3: Effect of Toremifene on ventral prostate development in theTRAMP mouse.

FIG. 4: Effect of Toremifene on tumor occurrence in the TRAMP mice.

FIG. 5: Effect of Toremifene on tumor development in the TRAMP model.

FIGS. 6A-6B: Comparison of placebo vs. Toremifene effects on tumorgrowth.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for preventing prostate;carcinogenesis; 2) methods for suppressing or inhibiting prostatecancer; 3) methods for reducing the risk of developing prostate cancer,and 4) methods for increasing the survival rate of a subject using anantiestrogen which does not cause the formation of DNA adducts as theprostate chemopreventive. In one embodiment the antiestrogen which doesnot cause the formation of DNA adducts is a chemopreventive agent havingthe formula:

wherein R₁ and R₂, which can be the same or different, are H or OH, R₃is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different,are H or an alkyl group of 1 to about 4 carbon atoms and analogs andmetabolites thereof; and their pharmaceutically acceptable carrier,diluents, salts, esters, or N-oxides, and mixtures thereof. Acomposition and/or a pharmaceutical composition may comprise thechemopreventive agent.

As demonstrated herein, toremifene is one example of an antiestrogenwhich does not cause the formation of DNA adducts which is a prostatechemopreventive agent. In the experiments conducted herein in bothanimal and human studies the antiestrogen was shown to prevent prostatecancer. The prostates were actually dissected and evaluated bothhistologically and by wholemount analysis. Also, tormifene was testedfor the prevention of prostate cancer by treating LNCaP xenografts innude mice. As is shown, the data is quite dramatic, not only has anantiestrogen such as toremifine inhibited growth, but actuallytoremifene was able to produce regression of the tumors. Further, inhuman studies conducted with the antiestrogen, high grade PIN (HGPIN)which has been established and time tested as a precursor lesion forhuman prostate cancer also know as latent prostate cancer, has shownregression. Thus, demonstrating that the antiestrogen toremifene is aprostate chemopreventive agent.

The present invention is directed to a method for preventing prostatecarcinogenesis. Antiestrogens which act as prostate chemopreventiveagents include but are not limited to: toremifene and analogs orsynthetics thereof: selective estrogen receptor modulators (SERMS),triphenylethylenes which include droloxifene, idoxifene,(2)-4-OH-tamoxifene; arzoxifene; chromans such as levomeloxifene, andcentchroman; benzothiophenes such as raloxifene, and LY 353381;naphthalens such as CP336,156; phytoestrogens such as lsoflavanoidsincluding daidzein, genistein, yenoestrogens; coumestrol; zearalenone;daidzein; apigenin; waempferol; phloratin; biochanin A; naringenin;formononetin; ipriflavone; quercetin; chrysin; flavonoids; flavones,isoflavones, flavanones, and chalcones): coumestans; mycoestrogens;resorcyclic scid lactone; nafoxideneand equol, and lignan includingenterodiol and enterolactone; and other compounds which are known in thead as follows: ICI 164,384, ICI 182, 780; TAT-59, EM-652 (SCG 57068),EM-800 (SCH57050), EM-139, EM-651, EM-776, and peptide antagonist ofhuman estrogen receptors.

This invention involves administering to a subject a pharmaceuticalpreparation of a chemopreventive agent having the formula:

wherein R₁ and R₃ which can be the sane or different, are H or OH, R₃ isOCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different, areH or an alkyl group of 1 to about 4 carbon atoms and analogs, andmetabolites thereof, and their pharmaceutically acceptable carrier,diluents, salts, esters, or N-oxides, and mixtures thereof.

This Invention provides the use of a pharmaceutical composition forpreventing prostate cancer, the recurrence of, suppression or inhibitionof prostate carcinogenesis, or increasing the survival rate of a subjecthaving prostate cancer, comprising an antiestrogen which does not causethe formation of DNA adducts and a suitable diluent. The antiestrogensinclude the antiestrogens provided above.

This invention provides for the use of a pharmaceutical composition forpreventing prostate cancer, the recurrence of, suppression or inhibitionof prostate carcinogenesis, or increasing the survival rate of a subjecthaving prostate cancer, comprising a chemopreventive agent having theformula:

wherein R₁ and R₂, which can be the same or different are H or OH R₃ isOCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different, areH or an alkyl group of 1 to about 4 carbon atoms; and theirpharmaceutically acceptable carrier, diluents, salts, esters, orN-oxides, land mixtures thereof.

The present invention provides a safe and effective method forpreventing carcinogenesis, suppressing or inhibiting latent prostatecancer and is particularly useful for treating subjects having anelevated risk of developing prostate cancer, for example, those havingbenign prostatic hyperplasia, prostate intraepithelial neoplasia (PIN),or an abnormally high level of circulating prostate specific antibody(PSA), or who have a family history of prostate cancer. In oneembodiment the subject is a mammalian subject. In another embodiment thesubject is a human subject.

The compound 4-chloro-1,2-diphenyl-1-[4-2-(N,N-dimethylamino)ethoxy]phenyl]-1-butene of formula (I), where R₁ and R₂ are each H andR₄ and R₅ are each methyl is named toremifene. Toremifene has been shownsafe and effective as an anti-tumor compound. On administration,toremifene has several metabolites that are also biologically active.

This Invention also provides for use of an antiestrogen which does notform DNA adducts and toremifene and analogs or metabolites thereof,which are well known to those skilled in the art. Other examples ofchemopreventive agents of formula (I) are the following:4-chloro-1,2-diphenyl-1-[4-[2-(N-methylamino)ethoxy]phenyl]-1-butene;4-chloro-1,2-diphenyl-1-[4-[2-(N,N-diethylamino)ethoxy]phenyl]-1-butene;4-chloro-1,2-diphenyl-1-[4-(aminoethoxy)phenyl1-butene;4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-2-phenyl]-1-butene;4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N-methylamino)ethoxy]phenyl]-2-phenyl-1-butene;and 4-chloro-1,2-bis(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-butene.

The invention encompasses pure (Z)- and (E)-isomers of the compounds andmixtures thereof as well as pure (RR,SS)- and (RS,SR)-enantiomer couplesand mixtures thereof.

The agent compounds of formula (I) can be prepared according toprocedures described in the previously cited U.S. Pat. Nos. 4,696,949and 5,491,173 to Toivola et al.

The invention includes pharmaceutically acceptable salts ofamino-substituted compounds with organic and inorganic acids, forexample, citric add and hydrochloric acid. The invention also includesN-oxides of the amino substituents of the compounds of formula (I).Pharmaceutically acceptable salts, can also be prepared from thephenolic compounds by treatment with inorganic bases, for example,sodium hydroxide. Also, esters of the phenolic compounds can be madewith aliphatic and aromatic carboxylic acids, for example, acetic acidand benzoic acid esters.

As used herein, pharmaceutical composition means therapeuticallyeffective amounts of the agent together with suitable diluents,preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A“therapeutically effective amount” as used herein refers to that amountwhich provides a therapeutic effect for a given condition andadministration regimen. Such compositions are liquids or lyphilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of In vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Other embodiments of thecompositions of the invention incorporate particulate forms protectivecoatings, protease inhibitors or permeation enhancers for various mutesof administration, including parenteral, pulmonary, nasal and oral. Inone embodiment the pharmaceutical composition is administeredparenterally, paracancerally, transmucosally, transdermally,intramuscularly, intravenously, intradermally, subcutaneously,intraperitonealy, intraventricularly, intracranially and intratumorally.The dosage may be in the range of 20-80 mg/day. In another embodimentthe dosage is in the range of 35-66 mg/day. In another embodiment thedosage is in the range of 40-60 mg/day. In another embodiment the dosageis in a range of 45-60 mg/day. The dosage may be 40-45 mg/day. Thedosage may be 60 mg/day. The dosage may be 45 mg/day.

Further, as used herein pharmaceutically acceptable carrier are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, collating agents, inert gasesand the like.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p,384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant will fail to elicit a humoral or cellular immuneresponse. Adjuvant include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gets such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvant such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral. Compounds modified by the covalent attachment of water-solublepolymers such as polyethylene glycol, copolymers of polyethylene glycoland polypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified compounds (Abuchowski et al., 1981;Newmark et al., 1982; and Katre et al., 1987). Such modifications mayalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. As a result, the desired in vivo biological activity may beachieved by We administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. For example, the agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng, 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment,polymeric materials can be used. In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984). Preferably, a controlled releasedevice is introduced into a subject in proximity of the site ofinappropriate immune activation or a tumor. Other controlled releasesystems are discussed in the review by Langer (Science 249:1527-1533(1990).

The method of the present invention for preventing prostatecarcinogenesis involves administering to a mammalian subject apharmaceutical preparation comprising chemopreventive agent or ametabolite or salt thereof. The pharmaceutical preparation can comprisethe chemopreventive agent alone, or can further include apharmaceutically acceptable carrier, and can be in solid or liquid formsuch as tablets, powders, capsules, pellets, solutions, suspensions,elixirs, emulsions, gels, creams, or suppositories, including rectal andurethral suppositories. Pharmaceutically acceptable carriers includegums, starches, sugars, cellulosic materials, and mixtures thereof. Thepharmaceutical preparation containing the chemopreventive agent can beadministered to a subject by, for example, subcutaneous implantation ofa pellet, in a further embodiment, the pellet provides for controlledrelease of chemopreventive agent over a period of time. The preparationcan also be administered by intravenous, intraarterial, or intramuscularinjection of a liquid preparation, oral administration of a liquid orsolid preparation, or by topical application. Administration can also beaccomplished by use of a rectal suppository or a urethral suppository.The pharmaceutical preparation can also be a parenteral formulation; inone embodiment, the formulation comprises a liposome that includes acomplex of a chemopreventive agent such as, for example, toremifene anda cyclodextrir compound, as described in the previously cited U.S. Pat.No. 5,571,534 to Jalonen et al.

The pharmaceutical preparations of the invention can be prepared byknown dissolving, mixing, granulating, or tablet-forming processes. Fororal administration, the chemopreventive agents or their physiologicallytolerated derivatives such as salts, esters, N-oxides, and the like aremixed with additives customary for this purpose, such as vehicles,stabilizers, or inert diluent, and converted by customary methods into 2suitable form for administration, such as tablets, coated tablets, hardor soft gelatin capsules, aqueous, alcoholic or oily solutions. Examplesof suitable inert vehicles are conventional tablet bases such aslactose, sucrose, or cornstarch in combination with binders like acacia,cornstarch, gelatin, or with disintegrating agents such as cornstarch,potato starch, alginic acid, of with a lubricant like stearic acid ormagnesium stearate. Examples of suitable oily vehicles or solvents arevegetable or animal oils such as sunflower oil or fish-liver oil.Preparations can be effected both as dry and as wet granules. Forparenteral administration (subcutaneous, intravenous, intraarterial, orintramuscular injection), the chemopreventive agents or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are converted Into a solution, suspension, or emulsion, ifdesired with the substances customary and suitable for this purpose, forexample, solubilizers or other auxiliaries. Examples are: sterileliquids such as water and oils, with or without the addition of asurfactant and other pharmaceutically acceptable adjuvant. Illustrativeoils are those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, or mineral oil. In general, water,saline, aqueous dextrose and related sugar solutions, and glycols suchas propylene glycols or polyethylene glycol are preferred liquidcarriers, particularly for injectable solutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino -ethanol,histidine, procaine, and the like.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the chemopreventive agents or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1633(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds,), Liss, N.Y., pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

The pharmaceutical compositions of the present invention areparticularly useful for treating a subject having an elevated risk ofdeveloping prostate cancer. High-risk subjects include, for example,those having benign prostatic hyperplasia, prostatic intraepithelialneoplasia (PIN), or an abnormally high level of circulating prostatespecific antibody (PSA), or who have a family history of prostatecancer.

Further, the prostate chemopreventive agent may be administered incombination With other cytokines or growth factors include but are notlimited to: IFN γ or α, IFN-β; interleukin (IL) 1, IL-2, IL-4, IL-6,IL-7, IL-12, tumor necrosis factor (TNF) α, TNF-β, granulocyte colonystimulating factor (G-CSF), granulocyte/macrophage CSF (GM-CSF);accessory molecules, including members of the integrin superfamily andmembers of the 19 superfamily such as, but not limited to, LFA-1, LFA-3,CD22, and B7-1, B7-2, and ICAM-1 T cell costimulatory molecules.

The chemopreventive agent may precede or follow a DNA damaging agenttreatment by intervals ranging from minutes to weeks. Protocols andmethods are known to those skilled in the art. DNA damaging agents orfactors are known to those skilled in the art and means any chemicalcompound or treatment method that Induces DNA damage when applied to acell. Such agents and factors include radiation and waves that induceDNA damage such as, gamma-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents”, function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide.

In another embodiment one may Irradiate the localized tumor site withDNA damaging radiation such as X-rays, UV-light, gamma-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with the DNAdamaging agent by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a DNAdamaging compound such as, adriamycin, 5-fluorouracil, etoposide,camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as gamma-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-Irradiation. It is most likely that all of these factors effect abroad range of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes.

As can be readily appreciated by one of ordinary skill in the art, themethods and pharmaceutical compositions of the present invention areparticularly suited to administration to a mammal, preferable a humansubject.

Intermediate endpoint biomarkers are measurable biologic alterations intissue that occur between the initiation of and the development of frankneoplasia. A biomarker is validated if the final endpoint, cancerincidence, were also reduced by the putative chemopreventive agent.Intermediate biomarkers in cancer may be classified into the followinggroups: histologic, proliferation, differentiation and biochemicalmarkers. In any chemoprevention strategy, the availability ofhistologically recognizable and accepted precancerous lesions constitutean important starting point For the prostate, a histological marker is aprecancerous precursor of prostatic adenocarcinoma of which prostaticintraepithelial neoplasia (PIN) is an example of. PIN appears as anabnormal proliferation within the prostatic ducts of premalignant fociof cellular dysplasia and carcinoma in situ without stromal invasion.PIN and histological prostate cancer are morphometrically andphenotypically similar. Thus, the development of high grade PINrepresents an important step in the progression pathway whereby thenormal prostate develops PIN, histological prostate cancer, invasiveclinical prostate cancer, and metastases.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXPERIMENTAL DETAILS SECTION EXAMPLE 1 Transgenic Adenocarcinoma MouseProstate

The study of prostate cancer chemoprevention has been hindered by thelack of appropriate animal models. The recent development of thetransgenic adenocarcinoma mouse prostate (TRAMP) model enables the studyof chemoprevention. In the TRAMP model, which is described in Greenberget al., A Prostate cancer in a transgenic mouse, @ Proc. Natl Acad. Sci.USA, 1995, Vol. 92, pages 3439-3443, the PB-SV40 large T antigen(PB-Tag) transgene is expressed specifically in the epithelial cells ofthe murine prostate. As a result, this model has several advantages overcurrently existing models: 1) mice develop progressive forms ofprostatic epithelial hyperplasia as early as 10 weeks and invasiveadenocarcinoma around 18 weeks of age; 2) the metastatic spread ofprostate cancer pattern mimics human prostate cancer with the commonsites of metastases being lymph node, lung, kidney, adrenal gland, andbone; 3) the development as well as the progression of prostate cancercan be followed within a relatively short period of 10-30 weeks; 4) thetumors arise with 100% frequency; and 5) the animals may be screened forthe presence of the prostate cancer transgene prior to the onset ofclinical prostate cancer to directly test treatment with chemopreventiveagents that may after prostate carcinogenesis.

The TRAMP transgenic mouse model is an excellent in vivo model todetermine the mechanisms of initiation and promotion of prostate cancerand to test the effectiveness of potential chemopreventive agents. Thesemice progressively develop prostate epithelial hyperplasia, PIN, andthen prostate cancer Within a short period (<17 weeks).

Chemopreventive treatment of hybrid TRAMP mice is initiated 30 dayspostnatally using chemopreventive agents at a level of about 0.5-50mg/kg of subject weight/day, preferably about 8-30 mg/kg of subjectweight/day. The chemopreventive agents are conveniently processed into21-day and 90-day pellets (prepared by Innovative Research of America,Sarasota, Fla.) and delivered as subcutaneous implants. Control animalsreceive placebo implants. In each drug treatment group, animals aresacrificed at 5, 7, 10, 15, 20, 25, 30, 40, and 50 weeks of age untilthe development of a palpable tumor. Blood is collected and pooled pertreatment time point to evaluate changes in serum testosterone andestradiol. Prostate tissues are harvested for morphometric, histologic,and molecular studies.

The following test procedures are employed:

1) Prostate wholemount analysis is serially performed to detect changesin prostate ductal morphology over time with and without treatment;examples are shown in FIG. 2. Tissue sections are evaluatedhistologically by H&E and Masson-trichome standard staining. Theemergence of PIN Is assessed and graded (I-mild to III-severe).

2) Serum estradiol and total testosterone levels are measured (RIA) foreach age interval to assess any changes in these hormones as a result ofchemopreventive agents.

EXAMPLE 2 Immunohistochemistry Data Analysis

Microscopy images of each tissue section are evaluated by usingcomputer-assisted (Mac 9500-132 computer and monitor) image quantitation(NIH-Image 1.6 PPC) using Kodak DCS 460 camera on Nikon Microphot-FXmicroscope and quantitated by using a color-assisted quantitative systemimage analysis (IPLab Spectrum 3.1, Scanalytics, Inc., Va.) thatdiscriminates color differences of stained tissue sections. Thresholdsare set to identify various tissue components of the prostate. The areapixel densities corresponding to each of these tissue components arecalculated for each full screen of the color monitor. A total of 5screens per prostate section are averaged. Immunohistochemical imagescan be digitalized and quantitated to enable statistical evaluation bydetermination of sample correlation coefficients and probability(2-tailed).

EXAMPLE 3 Study of Chemopreventive Activity

A study was undertaken to test the efficacy of chemopreventive agents inTRAMP transgenic animals (PSTag X FVBwt)(provided by Dr. NormanGreenberg, Baylor College of Medicine, Tex.). These mice showedpreliminary signs of cancer as early as 10 weeks. The TRAMP transgenicmale litters were screened for the Large T as transgene, and thepositive males were used in the study. The antiestrogen toremifene,which was to be tested for its possible chemopreventive effects, wasincorporated in customized pallets (Innovative Research of America,Sarasota, Fla.), and chemopreventive treatment of mice was initiatedpostnatally at 30 days (average mouse weight 14 g). Four groups of 10-12animals each received subcutaneous implantations of 90 day-releasetoremifene-containing pellets. The diffusible drug dosage, adjusted forgrowth related changes in weight, was designed to deliver either a lowdose (6 mg/kg) or a high dose (30 mg/kg) of toremifene Control animals(n=10) received placebo implants. The efficacy of the treatment wasmeasured by the absence of palpable tumor formation. The murine prostatetumors were harvested and evaluated by molecular and histologicaltechniques.

Using the TRAMP transgenic model of prostate cancer, in which everyanimal that inherits the prostate cancer gene develops prostate cancer,it was demonstrated that toremifene both increases the latency anddecreases the incidence of prostate cancer.

As shown in FIG. 1 the effects of low and high dose tormifene were botheffective. Tumor formation in the TRAMP mouse ventral prostate was notedat week 17 for the placebo group (n=10), at week 19 for the high dosetoremifene-treated group(n=12), and at week 28 for the low dosetoremifene-treated group (n=12). Thus, 5 treatment by toremifeneSubstantially increased the latency period by up to 11 weeks for thedevelopment of cancer in the ventral prostate of TRAMP mice.

Since the toremifene-treated animals did not reach the 50% tumordevelopment point during the period of the study, the time in which 25%of the animals had tumors was compared among groups. Tumors werepalpable in 25% of 10 the animals by week 23 in the placebo group and by30-31 weeks in the high and low toremifene groups, a delay of 7-8 weeks.Both low toremifene and high toremifene vs placebo were significant bylog rank and Wilcoxon statistical analysis, as shown in Table 1 below.

TABLE 1 Statistical Analysis Log-Rank Wilcoxon P p Low toremifene vsplacebo 0.0003* 0.0004* High toremifene vs placebo 0.0017* 0.0071**significance P < 0.05

At week 33, a point when all of the control animals had developedtumors, 72% of the low dose and 60% of the high dose toremifene-treatedanimals were still tumor-free. Thus, toremifene treatment at both lowand high dosages resulted in a greatly decreased incidence of tumors inthe ventral prostate of TRAMP mice.

These results, obtained in accordance with the present invention, wouldnot have been predicted from those reported In the aforementioned paperof Lucia et al., which describes the administering at two dosage levelsof tamoxifen, a close structural analog of toremifene, to Lobund-Wistarrats having prostate carcinomas induced by treatment with a combinationof an initiator and a promoter. In the Lucia et al. reference, it isreported that only 22-26% of the animals receiving the lower dose andonly 32-50% of those receiving the higher dose of tamoxifen remainedfree of tumors in the anterior prostate. It should be noted that theanterior prostate of a rodent, unlike its ventral prostate, has nocorresponding segment in the prostate of a human subject.

In Lucia et al., it is further stated that the initiator-promotercombination employed in the described procedures, although effective ininducing cancer in the anterior prostate of the test animals, failed toinduce carcinomas in the ventral prostate. Therefore there Is no basisto expect a chemopreventive effect on tumors in the ventral prostate byadministering tamoxifen, to Lobund-Wistar rats or to humans.

As already discussed, administering toremifene produces a substantialchemopreventive effect against tumors in the ventral prostate of TRAMPmice. This result is encouraging for a similar beneficial effect onhuman subjects, whose prostate does include a segment corresponding tothe ventral prostate of rodents.

EXAMPLE 4 Histological Examination of Prostate Tissue

Tumors from the placebo and high toremifene-treated groups taken at thetime of palpation were evaluated histologically. FIG. 2A is an H&Esection of the ventral prostate of a 17-week-old normal adult mouse.FIG. 2B, a section of the ventral prostate of a placebo-treated16-week-old TRAMP mouse, shows that, unlike the normal prostatestructure depicted in FIG. 2A, the TRAMP mouse ventral prostate ischaracterized by sheets of undifferentiated, anaplastic cells with ahigh mitotic index. In contrast, as shown in FIG. 2C, the prostate of atoremifene-treated 30-week-old TRAMP mouse retains much of the normalglandular architecture and ha tumors with a more differentiatedstructure, the mitotic index being much lower than that for theplacebo-treated animal. These results indicate that toremifene, even atlow dosage, is able to suppress prostate carcinogenesis in the TRAMPmodel.

EXAMPLE 5 Use of Chemopreventive Efficacy of Toremifene Against ProstateCancer in the TRAMP Mouse Model

This experiment confirms and demonstrates the chemopreventive efficacyof toremifene. This present study focuses on the histological andmolecular changes associated with development of prostate tumor incontrol animals and the mechanism of toremifene chemopreventive actionwith TRAMP animals which are bred, screened and treated withsustained-release drug pellets. At predetermined times, groups of 5animals were sacrificed and their prostates were removed for analysis.The prostate glands were evaluated for the presence of tumor byhistology, wholemount dissections, and large T antigenimmunohistochemistry. To date, the Placebo and the Toremifene treatmentshave been completed for the 7, 10, 15 and 20 week time-points and theresults are described below.

Result: Prostatic wholemounts for 7,10, 15, and 20 weeks for the variousgroups have been completed. Wholemount analysis revealed that placebotreated mice developed prostate tumors by 15-20 weeks of age similar tothe previous pilot study. Moreover, the Toremifene treated animals had adelay in the occurrence of prostate cancer up to 20 weeks (FIG. 3). By20 weeks, there is a striking delay in tumor occurrence in theToremifene treated group up to 35 weeks FIG. 4). These data confirm thateven with a more sensitive assessment of tumorigenicity. Toremifeneexhibited chemopreventive activity. For histological evaluation, tissuesamples were fixed, processed and paraffin embedded. Sections (5 pMthick) were cut and stained by routine H&E method. Toremifene inhibitedthe ductal development and tissue differentiation (compare the 17 weeksTRAMP mouse prostate tumor vs. wildtype (FIG. 4); b) Toremifene treatedprostate histology vs. Placebo at 15 weeks (FIG. 5) Qualitatively,immunohistochemistry of Placebo and Toremifene treated tissues showedpresence of T-antigen in the ventral prostate. Thus, the chemopreventiveactivity seen by Toremifene does not appear to be by suppression of theprobasin promoter in the TRAMP model.

Conclusions: The ability of Toremifene to prevent the occurrence ofprostate cancer in the TRAMP model has been confirmed utilizing moresensitive techniques to assess tumor formation. The mechanism ofToremifene=s chemopreventive effects does not appear to be through lossof the transgene for the Large T-antigen protein.

EXAMPLE 6 Toremifene Induces Regression of Established Human ProstateCancer Tumors in the Nude Mouse Model

Prostate cancer currently remains the most commonly diagnosed cancer inAmerican males. However, questions remain about the etiology andtreatment of this disease especially is advanced forms. Hormone therapyremains the standard method of treatment for recurrent and advancedprostate cancer despite the common development of hormone refractorydisease. Therefore, new approaches for the prevention and treatment ofprostate cancer are needed to accommodate the increasing number of mendiagnosed with this disease. The experiments and results belowdemonstrate that toremifene suppresses hormone sensitive LNCaP tumorgrowth in athymic nude mice.

Materials and Methods: One million LNCaP calls in Matrigel weresubcutaneously injected into each flank of athymic nude mice. A total 40mice were injected. After approximately 34 weeks, visible tumorsdeveloped. After recording the tumor size in two dimensions, the micewere divided into placebo and treatment groups based on equivalent tumorburden. A single pellet (placebo versus toremifene 35 mg) wassubcutaneously implanted between the scapulae of each mouse. Weeklymeasurements of the tumor size were recorded. Tumor volume wascalculated (tumor volume=0.5 (L+W)×L×W×0.5236, where L=tumor length andW=width). The tumor volume at the time of pellet implantation served asthe point of reference for future comparison of that tumor=s sizevariation. The weekly variations of each tumor volume were recorded aspercent differentiation from the original measurement at pelletimplantation.

Result: Two mice died soon after pellet implantation due to mortalwounds from other mice. One mouse treated with toremifene was excludedfrom the study due to excessive tumor hemorrhage and hematomadevelopment. All mice developed visible tumors unilaterally orbilaterally. Each tumor was followed independently for the duration ofthe study. Twenty-four tumors were treated with placebo and 28 tumorswere treated with toremifene. The results are shown in Table 2a andFIGS. 6A and 6B.

TABLE 2 Week N = % Change in volume relative to day 0 of treatmentPLACEBO GROUP 3 11  9.44 4 8 115.27 5 8 271.71 6 8 600.88 TOREMIFENE 311  −34.58 4 7 −61.01 5 7 −74.51 6 5 −61.72

The follow-up interval will be extended on the currently reportedpopulation and data on additional animals are presently being collected.

Conclusion: Toremifene inhibits and induces regression of establishedLNCaP tumors. Although the mechanism by which toremifene exerts thiseffect is unknown, the ability to produce these effects supports the useof Toremifene as a treatment for prostate cancer and to prevent therecurrence of prostate cancer in high risk patients with establishedprostate cancer micrometastases.

EXAMPLE 7 The Role of Antiestrogens: Tamoxifen citrate and Raloxifene(SERMs) and Faslodex (pure antiestrogen ICI 182,780) in the Preventionof Prostate Cancer

Experimental design: Chemopreventive treatment of mice are initiatedpost-natal at 30 days. Three groups of 50 hybrid TRAMP male mice eachare treated with either tamoxifen, citrate, or Raloxifene (SERMs) orFaslodex (pure antiestrogen ICI 182,780). The drugs is obtained ascustomized sustained-release pellets (Innovative Research of America,Sarasota, Fla.) and delivered as subcutaneous implants (see preliminarydata). Control animals are receive placebo implant with nopharmacological activity. Animals (n=10) are sacrificed at periodicintervals 10, 15, 20, 25 and 30 weeks age and the efficacy of thetreatment leading to either absence of tumor formation or reduction intumor size, if present, are assessed by comparison with placebo controlanimals. Blood is collected to evaluate changes in serum androgens andestrogens with each treatment. Prostatic tissues is saved for: a)morphometric studies; b) for histologic studies the tissue will be fixedin 10% buffered formalin, processed and paraffin embedded; c) formolecular studies the tissues is frozen in liquid nitrogen and stored at−70° C. Necropsies and survival data is also recorded.

The results of the experiment reveal the relative chemopreventiveefficacy of the various antiestrogens in the delay or prevention ofprostate cancer in the TRAMP model. The morphological studies Indicatethe gross changes, if any, in the development of the prostate size andductal pattern as a result of each treatment. Paraffinized tissuesections are stained using standard H&E techniques for histologicalchanges such as PIN that will be assessed to monitor the appearance ofprecancerous lesions as a precuror of prostatic adencarcinoma. Serumestradiol and total testosterone levels are measured for each ageinterval to assess any changes in these hormones, and whether or notthey correlate to changes in PIN. The peptide growth factor levels ofTGF, TGF 1, TGF 3, and bFGF is quantitative in prostate samples taken ateach interval. Corresponding peptide growth factor receptors is alsoassessed for EGFR and TGF RI and RII.

EXAMPLE 8 Toremifene Causes Regression of HGPIN in a Phase IIa ProstateCancer Chemoprevention Human Clinical Trial

The chemopreventive effects of an antiestrogen, toremifene againstprostate cancer have been reproducibly demonstrated herein in awell-established animal model of spontaneous human prostate cancer. Thisrepresents the first compound to demonstrate chemopreventive activityagainst prostate cancer. Moreover, High-grade PIN (HGPIN) has beenestablished and time tested as a precursor lesion for human prostatecancer also known as latent prostate cancer.

Consequently, HGPIN is used as an intermediate endpoint or surrogateendpoint for human prostate cancer. In fact, the NCI has now recommendedthat PIN should be used as an intermediate endpoint, of surrogateendpoint for human prostate cancer.

A Phase IIa, open labeled non randomized single center study with 17human subjects was conducted. In this protocol, patients with biopsyproven PIN are treated with 60 mg of Toremifene daily for 4 months.After 4 months, patients are rebiopsied and PIN status reassessed.Toremifene reduces PIN which thus will directly translate to a decreasein the incidence and a prolongation of the latency of prostate cancer.Other variables that are being investigated include serum PSA, qualityof life issues, and prostatic expression of TGFPβ1 expression (thepresumed mechanism of action).

17 patients had high grade PIN. HGPIN was determined from 8 biopsiesfrom biopsy session 1 which was confirmed 3 weeks later with repeat 8prostate biopsy cores from biopsy session 2. After 4 months ofToremifene treatment (60 mg/day), the patients were subjected toprostate biopsy session of 8 prostate core biopsies from the same areaspreviously biopsied (left and fight apex, midbase, base, and transitionzones).

The pathological evaluation revealed complete resolution of PIN withatrophic changes in the prostatic epithelum. The patient experienced noacute or chronic toxicites while taking Toremifene. The serum PSA, serumfree testosterone, serum total testosterone, and serum estradiolremained in the normal ranges. Quality of life was unchanged includingno affect on potency and libido. Therefore, these results demonstrate aprostate chemopreventive role for the antiestrogen toremifene.

What is claimed is:
 1. A method of suppressing or inhibiting pre-malignant lesions of prostate cancer of a subject by administering to the subject a pharmaceutical composition comprising an analog and/or a metabolite of a compound represented by the formula:

wherein R₁ and R₂, which can be the same or different, are H or OH; R₃ is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different, are H or an alkyl group of 1 to about 4 carbon atoms; and their pharmaceutically acceptable salts, esters, or N-oxides, and mixtures thereof.
 2. A method of treating a subject with pre-malignant lesions of prostate cancer by administering to the subject a pharmaceutical composition comprising an analog and/or a metabolite of a compound represented by the formula:

wherein R₁ and R₂, which can be the same or different, are H or OH; R₃ is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different are H or an alkyl group of 1 to about 4 carbon atoms; and their pharmaceutically acceptable salts, esters, or N-oxides, and mixtures thereof.
 3. A method of suppressing or inhibiting latent prostate cancer of a subject comprising the steps of: administering to the subject a pharmaceutical composition comprising an analog and/or a metabolite of a compound represented by formula:

wherein R₁ and R₂, which can be the same or different, are H or OH; R₃ is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different are H or an alkyl group of 1 to about 4 carbon atoms; and their pharmaceutically acceptable salts, esters, or N-oxides, and mixtures thereof.
 4. A method of treating a subject with prostate cancer comprising the steps of: administering to the subject a pharmaceutical composition comprising an analog and/or a metabolite of a compound represented by the formula:

wherein R₁ and R₂, which can be the same or different, are H or OH; R₃ is OCH₂CH₂NR₄R₅, wherein R₄ and R₅, which can be the same or different, are H or an alkyl group of 1 to about 4 carbon atoms; and their pharmaceutically acceptable salts, esters, or N-oxides, and mixtures thereof.
 5. The method of any of claims 1-2, wherein the pre-malignant lesion is a precancerous precursors of prostate adenocarcinoma.
 6. The method of claims 5, wherein the precancerous precursors of prostate adenocarcinoma is prostate intraepithelial neoplasia (PIN).
 7. The method of claim 6, wherein the prostate intraepithelial neoplasia is high prostate intraepithelial neoplasia (HPIN).
 8. The method according to any of claims 1, 2, 3, or 4, wherein said pharmaceutical composition further comprises an acceptable carrier or diluent.
 9. The method according to claim 8, wherein said carrier is selected from the group consisting of a gum, a starch, a sugar, a cellulosic material, or mixtures thereof.
 10. The method according to claim 8, wherein said antiestrogen is administered subcutaneously, orally, intravenously, intraarterially, intramuscularly, or topically.
 11. The method according to claim 8, whereby said subcutaneous administration is by implanting in said subject a pellet containing said pharmaceutical composition.
 12. The method according to claim 11, wherein said pellet provides for controlled release of said pharmaceutical preparation over a period of time.
 13. The method according to claim 8, whereby said intravenous, intra-arterial, or intramuscular administration is by intravenously, intraarterially, or intramuscularly injecting in said subject said pharmaceutical composition in a liquid form.
 14. The method according to claim 8, whereby said oral administration is by orally administering to said subject in a liquid or solid preparation containing said pharmaceutical composition.
 15. The method according to claim 8, whereby said topical administration is by applying to skin surface of said subject said pharmaceutical composition.
 16. The method according to claim 15, wherein said pharmaceutical composition is selected from the group consisting of a pellet, a tablet a capsule, a solution, a suspension, an emulsion, an elixir, a gel, a cream, and a suppository.
 17. The method according to claim 16, wherein said suppository is a rectal suppository or a urethral suppository.
 18. The method according to claim 8, wherein said pharmaceutical composition is a parenteral formulation.
 19. The method according to claim 18, wherein said parenteral formulation comprises a liposome.
 20. The method of claims 1, 2, 3, or 4, wherein the analog is, 4-chloro-1,2-diphenyl-1-[4-[2-(N-methylamino)ethoxy]phenyl]-1-butene; 4-chloro-1,2-diphenyl-1-[4-[2-(N,N-diethylamino)ethoxy]phenyl]-1-butene; 4-chloro-1,2-diphenyl-1-[4-(aminoethoxy)phenyl]-1-butene; 4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-2-phenyl-1-butene; 4-chloro-1-(4-hydroxyphenyl)-1-[4-[2-(N-methylamino)ethoxy]phenyl]-2-phenyl-1-butene; or 4-chloro-1,2-bis(4-hydroxyphenyl)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-butene. 